Proposal Summary Facial palsy (FP) is a vexing clinical condition with massive functional, aesthetic, and communication sequelae. The gold-standard clinical approach to smile reanimation in FP involves placing a nerve autograft across the upper lip that routes axons from a healthy-side facial nerve donor branch to the paralyzed side. In a subsequent surgery, free muscle is transplanted into the paralyzed side and neurotized by the cross-facial nerve graft (CFNG). The operation carries a 30% failure rate for incompletely understood reasons. Emerging evidence suggests Schwann cells (SCs) in distal portions of long grafts become less supportive of axonal regeneration following prolonged periods without axonal contact. Axonal growth arrest across long grafts results in suboptimal neurotization of distal targets and procedural failure. The molecular mechanisms underlying SC response to long-term denervation remain incompletely understood. Prolonging the pro- regenerative SC state within nerve grafts is a promising therapeutic target for enhancing clinical outcomes in smile reanimation. The goal of the proposed research is to characterize the transcriptional responses of CFNG SCs and facial motor neurons (FMNs) following axotomy and coaptation to freshly harvested as compared to long-term denervated nerve grafts. While prior work has employed quantitative PCR and bulk RNA sequencing to study peripheral nerve regeneration, such an approach assumes SCs and neurons are homogeneous populations. Herein, we hypothesize that transcriptional heterogeneity exists among nerve graft SCs following injury, and that differences exist between transcriptomes of FMNs whose axons are coapted to freshly harvested as opposed to long-term denervated nerve grafts. A novel mouse surgical model of prolonged nerve graft denervation simulating long nerve grafts in humans will be developed, and relationships between SC and FMN expression assessed using DroNc-seq, a novel massively parallel single nucleus RNA-sequencing technique. New knowledge will be gained regarding the molecular mechanisms underlying SC and FMN transcriptional changes following injury and nerve grafting. Such knowledge could inform therapeutic targets to prolong the SC repair state, mitigate apoptosis, and enhance clinical outcomes in nerve regeneration and smile reanimation.